A. Field of the Invention
This invention relates to recombinant DNA vectors useful for producing recombinant baculovirus or stably-transformed insect cell lines.
B. Description of the Related Art
Baculovirus expression vectors (BEVs) have become extremely important tools for the expression of foreign genes, both for basic research and for the production of proteins with direct clinical applications in human and veterinary medicine (W. Doerfler, Curr. Top. Microbiol. Immunol., 131:51-68 (1968); V. A. Luckow and M. D. Summers, Bio/Technology. 6:47-55 (1988a); L. K. Miller, Annual Review of Microbiol., 42:177-199 (1988); M. D. Summers, Curr. Communications in Molecular Biology, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1988)). BEVs are baculovirus recombinant insect vectors in which the coding sequence for a chosen foreign gene has been inserted behind the promoter in place of the nonessential viral gene, polyhedrin (Smith and Summers, U.S. Pat. No., 4,745,051).
Baculovirus genes are expressed in a sequential, temporally-regulated fashion during one or more of four different phases of the viral replication cycle (P. D. Friesen and L. K. Miller, Curr. Top. Microbiol. Immunol., 131:31-49 (1986); L. A. Guarino, CRC Press, (1989) [in press]). Therefore, different baculovirus genes may be classified as immediate-early (.alpha.), delayed-early (.beta.), late (.gamma.), or very late (.delta.), according to the phase of the viral infection during which they are expressed.
The expression of these genes occurs sequentially, probably as the result of a "cascade" mechanism of transcriptional regulation. Thus, the immediate-early genes are expressed immediately after infection, in the absence of other viral functions, and one or more of the resulting gene products induces transcription of the delayed-early genes. Some delayed-early gene products, in turn, induce transcription of late genes, and finally, the very late genes are expressed under the control of previously expressed gene products from one or more of the earlier classes. One relatively well-defined component of this regulatory cascade is IE1, an immediate-early gene of Autographa californica nuclear polyhedrosis virus (AcMNPV). IE1 is expressed in the absence of other viral functions and encodes a product that stimulates the transcription of several genes of the delayed-early class, including the 39K gene (L. A. Guarino and M. D. Summers, J. Virol., 57:563-571 (1986a); J. Virol., 61:209-2099 (1987)), as well as late genes (L. A. Guarino and M. D. Summers, Virol., 162:444-451 (1988)). However it is believed that the immediate-early genes are not dependent upon other viral genes for expression.
The polyhedrin gene is classified as a very late gene. Therefore, transcription from the polyhedrin promoter requires the previous expression of an unknown, but probably large number of other viral and cellular gene products. Because of this, state-of-the-art BEVs, such as the exemplary BEV system described by Smith and Summers (U.S. Pat. No., 4,745,051) will express foreign genes only as a result of gene expression from the rest of the viral genome, and only after the viral infection is well underway. In addition, this vector is not suitable for expression in live insects because it cannot produce occluded viral progeny.
The expression of foreign genes as a result of gene expression from the rest of the viral genome represents a clear limitation to the use of the traditional BEV system for at least two reasons. First, infection with the essentially intact recombinant virus ultimately kills the host cell, thereby terminating its role as a "factory" for foreign protein production. Therefore, disadvantages to the expression of foreign genes under the control of baculovirus "very late" promoters (polyhedrin and p10 genes) are that expression from these promoters can only occur in insect cells infected with a recombinant baculovirus during the very late phase of the infection. Because these host cells die after 4-5 days of infection, expression with these vectors is what is defined as transient. Thus, prior art BEV systems are limited to transient gene expression in cell lines.
The second limitation is the decrease in the ability of the host cell to process newly synthesized proteins as the baculovirus infection progresses (D. L. Jarvis and M. D. Summers, Mol. Cell. Biol., 9:214-223 (1989)). After only one day of infection, the infected cells no longer process foreign glycoproteins efficiently, as previously shown for human tissue plasminogen activator. Thus, gene expression from the polyhedrin promoter occurs at a time when the host cell's ability to process newly synthesized proteins is significantly diminished.
Thus, there is a great need for a system that expresses foreign gene products in a continuous and permanent manner such that the cell is still capable of processing glycoprotein products efficiently. Described herein is such a system, as well as an improved and novel vector for gene expression employing baculovirus early promoters.
The novel and improved plasmid vector described in this invention can be used to produce either insect cell transformants or recombinant baculoviruses. This vector expresses a foreign gene either in uninfected insect cells or during the immediate early phase of infection in insect cells or in insects found in nature. Thus, this plasmid vector is suitable for expression in live insects because it can be incorporated into recombinant viruses that can produce occluded viral progeny. This novel plasmid vector retains a functional polyhedrin gene which is necessary to stabilize the virus against environmental inactivation.
This also allows the recombinant viruses produced with these plasmid vectors to produce occluded progeny. These progeny are highly infectious in vivo. This is not true for other recombinant baculoviruses, which produce only non-occluded progeny. Thus, the new vectors will have the ability to express foreign genes in vivo and expression will occur during the early stages of infection. These properties are particularly critical for the expression of foreign gene products, including insecticides, in insects. Together, these features make this vector ideal for use in environmental applications particularly for pest control.
This invention has direct utility in terms of effectively disseminating potent insecticides to the environment. One of the novel aspects of this invention is the use of early baculovirus gene expression for effective insecticide delivery. Insect viral infections, because of a late baculovirus promoter, normally take 3-5 days to kill the insect. During this time the insects will continue to feed on the crop. In 3-5 short days, insects infected with viruses can devastate the crops and the farmer's future. Thus, by using insecticides under the direct control of promoters derived from early baculovirus genes, insects will be killed or at least prevented from continuing to feed on the crops. Death of the insect or prevention from further feeding should occur within hours of ingesting viruses.
Another advantage for baculovirus expression vector system users is infected cells are healthier while still producing the desired foreign genes.